Nanoparticles and microparticles of non-linear hydrophilic-hydrophobic multiblock copolymers

ABSTRACT

Injectable particles are provided that are not rapidly cleared from the blood stream by the macrophages of the reticuloendothelial system, and that can be modified as necessary to achieve variable release rates or to target specific cells or organs as desired. The injectable particles can include magnetic particles or radiopaque materials for diagnostic imaging, biologically active molecules to be delivered to a site, or compounds for targeting the particles. Biodistribution experiments indicate that the injectable particles have a prolonged half-life in the blood compared to particles not containing poly(alkylene glycol) moieties on the surface.

This invention was made with government support under Grant NumberNIH-1R01-GM44884 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

The present application is a continuation-in-part of U.S. Ser. No.08/210,677, "Biodegradable Injectable Particles for Imaging," filed Mar.18, 1994, by Ruxandra Gref, Yoshiharu Minamitake and Robert S. Langer,which is a continuation-in-part of U.S. Ser. No. 08/096,370,"Biodegradable Microparticles and Injectable Nanoparticles" filed Jul.23, 1993, by Ruxandra Gref. Yoshiharu Minamitake and Robert S. Langer,the contents of which are hereby incorporated by reference.

This invention is in the area of biodegradable block copolymers andinjectable nanoparticles and microparticles for the controlled deliveryof biologically active materials and diagnostic purposes made from thepolymers.

BACKGROUND OF THE INVENTION

A major challenge in the area of the parenteral administration ofbiologically active materials is the development of a controlleddelivery device that is small enough for intravenous application andwhich has a long circulating half-life. Biologically active materialsadministered in such a controlled fashion into tissue or blood areexpected to exhibit decreased toxic side effects compared to when thematerials are injected in the form of a solution, and may reducedegradation of sensitive compounds in the plasma.

A number of injectable drug delivery systems have been investigated,including microcapsules, microparticles, liposomes and emulsions. Asignificant obstacle to the use of these injectable drug deliverymaterials is the rapid clearance of the materials from the blood streamby the macrophages of the reticuloendothelial system (RES). For example,polystyrene particles as small as sixty nanometers in diameter arecleared from the blood within two to three minutes. By coating theseparticles with block copolymers based on poly(ethylene glycol) andpoly(propylene glycol), their half-lives were significantly increased.L. Illum, S. S. Davis, "The organ uptake of intravenously administeredcolloidal particles can be altered by using a non-ionic surfactant(poloxamer 338)", FEBS Lett., 167, 79 (1984).

Liposomal drug delivery systems have been extensively considered for theintravenous administration of biologically active materials, becausethey were expected to freely circulate in the blood. It was found,however, that liposomes are quickly cleared from the blood by uptakethrough the reticuloendothelial system. The coating of liposomes withpoly(ethylene glycol) increases their half life substantially. Theflexible and relatively hydrophilic PEG chains apparently induce astearic effect at the surface of the liposome that reduces proteinadsorption and thus RES uptake. T. M. Allen, C. Hansen, Biochimica etBiophysica Acta, 1068, 133-141 (1991); T. M. Allen, et al., Biochimicaet Biophysica Acta, 1066, 29-36 (1991); V. Torchilin, A. Klibanov, "TheAntibody-linked Chelating Polymers for Nuclear Therapy and Diagnostics",Critical Reviews in Therapeutic Drug Carrier Systems, 7(4), 275-307(1991); K. Maruyama, et al., Chem. Pharm. Bull., 39(6), 1620-1622(1991); M. C. Woodle, et al., Biochimica et Biophysica Acta; 193-200(1992); and D. D. Lassic, et al., Biochimica et Biophysica Acta, 1070,187-192 (1991); and A. Klibanov, et al., Biochimica et Biophysica Acta,1062, 142-148 (1991).

European Patent Application Nos. 0 520 888 A1 and 0 520 889 A1 disclosenanoparticles made from linear block copolymer of polylactic acid andpoly(ethylene glycol) for the injectable controlled administration ofbiologically active materials. The applications do not disclose how tomodify the copolymer to vary the profile of drug release or howmodifying the copolymer would affect distribution and clearance of thedelivery devices in vivo. The applications also do not teach how toprepare nanoparticles that are targeted to specific cells or organs, orhow to prepare nanospheres that are useful for gamma-imaging fordiagnostic purposes.

In U.S. Ser. No. 08/690,370 filed Jul. 23, 1993, injectable particlesare described which are formed of a biodegradable solid core containinga biologically active material and poly(alkylene glycol) moieties on thesurface or of block copolymers of the poly(alkylene glycol) moietieswith biodegradable polymers, which exhibit increased resistance touptake by the reticuloendothelial system.

It would be desirable to have other types of injectable particles forthe controlled delivery of materials that are not rapidly cleared fromthe blood stream by the macrophages of the reticuloendothelial system,and that can be modified as necessary to target specific cells or organsor manipulate the rate of delivery of the material.

It is an object of the present invention to provide copolymers forpreparing microparticles or nanoparticles or coatings which decreaseuptake by the reticuloendothelial system and are readily derivatized.

It is another object of the present invention to provide injectableparticles for the controlled delivery of diagnostic and therapeuticmaterials that are not rapidly cleared from the blood stream.

It is another object of the present invention to provide injectablemicroparticles or nanoparticles that can be modified as necessary totarget specific cells or organs or manipulate the rate of delivery ofthe material.

It is another object of the present invention to provide injectablebiodegradable microparticles or nanoparticles that contain detectablematerials for diagnostic imaging.

SUMMARY OF THE INVENTION

Non-linear multiblock copolymers are prepared by covalently linking amultifunctional compound with one or more hydrophilic polymers and oneor more hydrophobic bioerodible polymers to form a polymer including atleast three polymeric blocks. In one embodiment, one or more hydrophilicpolymers, such as polyethylene glycol (PEG) chains or polysaccharidemoieties, are covalently attached to a multifunctional molecule such ascitric acid or tartaric acid, leaving one or more active hydroxyl,carboxylic acid or other reactive functional groups available to attachthe hydrophobic polymer(s). The hydrophobic polymer, such as polylacticacid (PLA), polyglycolic acid (PGA), polyanhydrides, polyphosphazenes orpolycaprolactone (PCL), is then covalently linked to the multifunctionalcompound via an appropriate reaction such as ring opening orcondensation polymerization. In one embodiment, the multiblockcopolymers can have several short PEG chains, for example, with amolecular weight less than 1000, attached to the multifunctionalcompound. Ligands can be attached to one or more polymer chains toachieve a variety of properties for a wide range of applications.

The coblock polymers are useful in forming coatings on implantabledevices and, in the most preferred embodiment, injectable nanoparticlesand microparticles that are not rapidly cleared from the blood stream bythe macrophages of the reticuloendothelial system, and that can bemodified as necessary to achieve variable release rates or to targetspecific cells or organs as desired. The particles can incorporatewithin or on their surface a substance to be delivered for eithertherapeutic or diagnostic purposes. In a preferred embodiment, thehydrophilic polymer is a poly(alkylene glycol) (PAG). The terminalhydroxyl group of the poly(alkylene glycol) or other hydrophilicpolymers can be used to covalently attach molecules onto the surface ofthe injectable particles. Materials incorporated onto or within theparticles include biologically active molecules and targeting moleculessuch as antibodies immunoreactive with specific cells or organs,compounds specifically reactive with a cell surface component, magneticparticles, detectable materials such as radiopaque materials fordiagnostic imaging, other substances detectable by x-ray or ultrasoundsuch as air, fluorescence, magnetic resonance imaging, and moleculesaffecting the charge, lipophilicity or hydrophilicity of the particle.

The typical size of the particles is between approximately 50 nm and1000 nm, preferably between 90 nm and 200 nm, although microparticlescan also be formed as described herein. The particles can beadministered by a variety of ways, although a preferred embodiment is byintravenous administration. The injectable particles are easilylyophilized and redispersed in aqueous solutions. Biodistributionexperiments indicate that the injectable particles have a prolongedhalf-life in the blood compared to particles not containingpoly(alkylene glycol) moieties on the surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a, 1b, and 1c are schematic representations of nanospheres formedof multiblock copolymers made by covalently linking a multifunctionalcompound with one or more hydrophilic polymers and one or morehydrophobic bioerodible polymers.

FIG. 2a is a schematic illustration of the synthesis of (PEG)₃-citrate-polylactide, (PEG)₃ -citrate-polycaprolactone and (PEG)₃-citrate-polysebacic acid, in which the polyethylene glycol blocks canbe functionalized with a ligand.

FIG. 2b is a schematic illustration of multiblock copolymers of tartaricacid and mucic acid with polylactic acid (PLA), polycaprolactone (PCL),polysebacic acid (PSA) and polyglycolic acid (PGA) hydrophobic blocks,and polyethylene glycol (PEG) hydrophilic blocks.

FIG. 2c is a schematic illustration of PEG-di-PLA.

FIG. 2d is a schematic illustration of multiblock copolymers of benzenetetracarboxylic acid with polyethylene glycol (PEG) and polylactic acid(PLA) or polysebacic anhydride (PSA) blocks.

FIG. 2e is a schematic illustration of the synthesis of butanediglycidyl ether-based tetra-arm diblock copolymers with polylactic acid(PLA) and polyethylene glycol (PEG) blocks.

FIG. 2f is a schematic illustration of multiblock copolymers of the1,4-3,6-dilactone of glucaric acid with ligand, polylactic acid (PLA)and polyethylene glycol (PEG) blocks.

FIG. 2g is a schematic illustration of (PEG)₃ -citrate-polylactide inwhich the PEG blocks are further functionalized with a ligand or PLA.

FIG. 2h is a schematic illustration of PLA-citrate-dextran andPLA-2-hydroxyadipaldehyde-Dextran.

FIG. 2i is a schematic illustration of PEG 2-hydroxyadipaldehyde-PLA inwhich the PEG can be functionalized with a ligand or a methyl group.

The non-linear block copolymers in each of FIGS. 2a through 2i weresynthesized from poly(ethylene glycol) [PEG] of the molecular weights600, 1900, 5,000; 12,000; and 20,000, and polylactide (PLA),polyglycolide, polycaprolactone (PCL), or polysebacic anhydride (PSA).

DETAILED DESCRIPTION OF THE INVENTION

Non-linear multiblock copolymers are prepared by covalently linking amultifunctional compound with one or more hydrophilic polymers and oneor more hydrophobic bioerodible polymers to form a polymer including atleast three polymeric blocks. In one embodiment, one or more hydrophilicpolymers, such as polyethylene glycol (PEG) chains or polysaccharidemoieties, are covalently attached to a multifunctional molecule such ascitric acid or tartaric acid, leaving one or more active hydroxyl,carboxylic acid or other reactive functional groups available to attachthe hydrophobic polymer(s). The hydrophobic polymer, such as polylacticacid (PLA), polyglycolic acid (PGA), polyanhydrides, polyphosphazenes orpolycaprolactone (PCL), is then covalently linked to the multifunctionalcompound via an appropriate reaction such as ring opening orcondensation polymerization.

Injectable particles formed of the coblock polymers are disclosed thatare not rapidly cleared from the blood stream by the macrophages of thereticuloendothelial system as the particles not surface modified withhydrophilic polymers, and that can be modified as necessary to achievevariable release rates or to target specific cells or organs as desired.The particles are useful to administer biologically active materials ina controlled manner for a wide variety of purposes.

I. Non-linear Block copolymers. Selection of Polymers.

Hydrophilic Polymers

Hydrophilic polymers, including but not limited to poly(alkyleneglycols) (which can also be referred to as a poly(alkylene oxide), ifthe polymer was prepared from an oxide instead of a glycol) andpolysaccharides, are employed as the hydrophilic portion of themultiblock copolymer. Hydrophilic polymers other than poly(alkyleneglycol) that can be used include polypyrrolidone, dextrans, andpoly(vinyl alcohol). Other materials include a Pluronic™ F68 (BASFCorporation), a copolymer of polyoxyethylene and polyoxypropylene, whichis approved by the U.S. Food and Drug Administration (FDA).

As used herein, the term poly(alkylene glycol) refers to a polymer ofthe formula HO--[(alkyl)O]_(y) --OH, wherein alkyl refers to a C₁ to C₄straight or branched chain alkyl moiety, including but not limited tomethyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Y is an integergreater than 4, and typically between 8 and 500, and more preferablybetween 40 and 500.

In vivo results show that the higher the molecular weight (MW) of PEG,the longer the circulation time in the blood (the half-life).

Specific examples of poly(alkylene glycols) include poly(ethyleneglycol), polypropylene 1,2-glycol, poly(propylene oxide) andpolypropylene 1,3-glycol. A preferred hydrophilic polymeric moiety isPEG of a molecular weight of approximately 500 to 20,000 molecularweight.

To ensure elimination from the body, the hydrophilic polymer, forexample, non-degradable PEG, should have a molecular weight ofapproximately 20,000 Daltons or less.

Hydrophobic Polymers

The hydrophobic polymer should be bioerodible, biocompatible, and have aterminal group that can react with the terminal functional group, suchas a hydroxyl, thiol, amino, carboxy, aldehyde or other functional groupof the multifunctional molecule to form a covalent linkage. Multiblockcopolymers containing polylactic acid moieties are a preferredembodiment. However, the copolymer of lactic acid and glycolic acid, aswell as other polymers such as polysiloxanes, polyanhydrides,polyphosphazenes, polymers of α-hydroxy carboxylic acids,polyhydroxybutyric acid, polyorthoesters, polycaprolactone, orcopolymers prepared from the monomers of these polymers can be used toform the multiblock copolymers described herein. The variety ofmaterials that can be used to prepare the block copolymers forming theinjectable particles significantly increases the diversity of releaserate and profile of release that can be accomplished in vivo.

In one embodiment, a hydrophobic polyanhydride to form the multiblockcopolymer. Biodegradable polyanhydrides are disclosed in, for example,U.S. Pat. Nos. 4,757,128, 4,857,311, 4,888,176, and 4,789,724.Polyhydroxybutyrates are disclosed in Agostini, S., "Synthesis andCharacterization of PHB," Ph.D. thesis, Case Western University, U.S.A.(1971) and U.S. Pat. No. 3,044,942. The teachings of these publicationsare incorporated by reference herein.

In a preferred embodiment, a polyester of poly(lactic-co-glycolic)acid(PLGA) is used as a hydrophobic erodible polymer bound to themultifunctional compound. These polymers are approved for parenteraladministration by the FDA. Because PLGA degrades via hydrolysis, in vivodegradation rates can be predicted from in vitro data. PLGA degrades tolactic and glycolic acids, substances found naturally in the body.Furthermore, by manipulating the molar ratio of lactic and glycolic acidand the molecular weight of the copolymers, different degradationpatterns can be obtained.

The molecular weight and the chemical composition and stereochemicalconfiguration of the polymer will affect the solubility of the polymerin various organic solvents as well as the crystallinity of the polymer.In this regard, a copolymer of lactic acid and glycolic acid ispreferable.

Preferably, the hydrophobic, bioerodible polymers are soluble in ethylacetate or acetone. Ethyl acetate or acetone is preferred over otherorganic solvents such as dichloromethane and chloroform because they areless toxic for in vivo applications.

Poly L-lactide is a polymer with a high degree of crystallinity. PolyD,L-lactide is less crystalline and more soluble in organic solvents. Arandom copolymer of D,L-lactide and glycolide in the ratio of 75:25 isvery soluble in organic solvents, in particular in ethyl acetate. Thiscopolymer is completely amorphous, which renders it a useful polymer forthe fabrication of nanospheres and microspheres for controlled release.

Poly-L-lactide has a degradation time in vitro of months to years. Thelong degradation time is due to its higher crystallinity which protectsthe polymer from water penetration. Since D,L-lactide is amorphous, itsdegradation time is typically one to a number of months. Poly-glycolidealso has a crystalline structure and a degradation time of one toseveral months. D,L-PLGA is amorphous, with a degradation time in vitroof weeks to months. As the glycolic acid ratio is increased, the rate ofdegradation is enhanced. Lactic acid has bulky methyl groups on thealpha carbon (--O--CH(CH₃ --CH--) which makes it difficult for watermolecules to access the ester, while glycolic acid has a proton on thealpha carbon (--O--CH₂ --CO--), which allows easier access of watermolecules to the ester bonds.

The molecular weight of the hydrophilic and hydrophobic regions of theinjectable particle affect the water solubility of the particles andthus their stability in aqueous solutions.

Preparation of multiblock copolymers.

The multiblock copolymers formed by covalently linking a multifunctionalcompound with one or more hydrophilic polymers, preferably poly(alkyleneglycol) (PAG), more preferably poly(ethylene glycol), and one or morehydrophobic polymers can be prepared by a number of methods. One methodinvolves protecting one end of the hydrophilic polymer, for example,polyethylene glycol, and reacting the functional group at theunprotected end with one or more reactive groups on the multifunctionalcompound. Then, the remaining reactive groups on the multifunctionalcompound can be reacted with one or more hydrophobic bioerodiblepolymers, followed by removal of the protecting groups. Selectiveremoval of the protecting groups allows selective modification of thehydrophobic and hydrophilic polymers, and is well known to those skilledin the art of polymer synthesis.

Preferred protected polyalkylene glycols include monomethoxypoly(alkylene glycols), such as monomethoxy-PEG or PEG protected withanother oxygen protecting group known to those of skill in the art, suchthat one terminal hydroxyl group is protected and the other is free toreact with the polymer.

A second method involves reacting a hydrophobic bioerodible polymer,with one terminal functional group protected, with one or more reactivegroups on the multifunctional compound, and then reacting a protectedhydrophilic polymer with one or more reactive groups remaining on themultifunctional compound.

In an alternative embodiment, a carboxylic acid group on themultifunctional compound can be reacted with a poly(alkylene glycol)terminated with an amino function (available from Sherwater Polymers,Inc.) to form an amide linkage, which is in general stronger than anester linkage. The amide linkage may provide a longer period ofretention of the poly(alkylene glycol) on the surface of thenanoparticle. Methods of linking amino groups with carboxylic acidgroups to form amides are well known to those skilled in the art.

In another alternative embodiment, a thiol group on a polymer can bereacted with a carboxy group on the multifunctional compound to form athioester linkage. Methods of forming thioester linkages are known tothose skilled in the art.

In yet another alternative embodiment, amino groups on a polymer can becoupled with amino groups on a multifunctional compound using acrosslinking agent such as glutaraldehyde. These coupling reactions areknown to those skilled in the art.

Other multiblock copolymers terminated with poly(alkylene glycol), andin particular, poly(ethylene glycol), can be prepared using thereactions described above, using a branched or other suitablepoly(alkylene glycol) and protecting the terminal groups that are not tobe reacted. Shearwater Polymers, Inc., provides a wide variety ofpoly(alkylene glycol) derivatives.

In one embodiment, a multiblock copolymer is prepared by reacting theterminal group of the hydrophobic polymeric moiety such as PLA or PLGAwith a suitable polycarboxylic acid monomer, including but not limitedto 1,3,5-benzenetricarboxylic acid, butane-1,1,4-tricarboxylic acid,tricarballylic acid (propane-1,2,3-tricarboxylic acid), andbutane-1,2,3,4-tetracarboxylic acid, wherein the carboxylic acidmoieties not intended for reaction are protected by means known to thoseskilled in the art. The protecting groups are then removed, and theremaining carboxylic acid groups reacted with a hydrophilic polymer,such as a poly(alkylene glycol). In another alternative embodiment, adi, tri, or polyamine is similarly used as the branching agent.

II. Preparation of Particles from Block Copolymers

Preparation and Characterization of Nanoparticles

Nanospheres can be prepared from the block copolymers byemulsion/evaporation techniques using the pre-formed copolymer. Thepre-formed polymer and, optionally, a substance to be delivered, ifsoluble in an organic solvent, can be dissolved in an organic solvent.Loadings can be about 25 mg polymer/2 ml methylene chloride, and thesubstance to be delivered in approximately between 10% and 50% of theweight of the polymer. The resulting organic solution can be emulsifiedwith an aqueous phase by vortexing and then sonicated, typically for 1minute, at 40-W output. The solvent can be evaporated and thenanospheres can be collected by centrifugation (30 min, 5,000 rpm),washed twice and lyophilized.

Amphiphilic multiblock copolymers can form nanospheres with abiodegradable and dense core able to entrap drugs or other compounds,and with an effective coating to prevent the rapid recognition by theimmune system. The different solubilities of the hydrophilic andhydrophobic blocks, for example, PEG and a polyester or polyanhydride,in water and organic solvents allows one to obtain the desiredphase-separated structure of the nanospheres. The organic phase,containing polymer and drug, can be emulsified with water without addingany further stabilizer, because of the surfactant properties of themultiblock copolymer. By emulsifying the two phases, the hydrophilicblock migrates to the water interface, and the hydrophobic block remainsinside the droplets and forms the solid biodegradable core after solventevaporation. Sub-200 nm size particles with a high PEG density on thesurface can be obtained using a high energy form such as ultrasound. AFManalysis indicates that nanospheres prepared in this manner arespherical, and QELS showed that the particle size of nanospheresprepared in this manner are in the range of 180-240 nm and have aunimodal size distribution.

For example, the mixture of block copolymer and substance to bedelivered can be mixed in a common solvent such as ethyl acetate ormethylene chloride. Preferably, the organic solvent is a nonsolvent forthe hydrophilic polymers, and a solvent for the hydrophobic polymers. Anemulsion can be formed by adding water, preferably distilled deionizedwater, to the solution. Slow evaporation of the organic solvent allows areorganization of the polymer chains inside and on the surface of thedroplets. The hydrophilic polymers, which are preferably insoluble inthe organic solvent, tend to migrate to the aqueous phase, while thehydrophobic polymers, which are not soluble in water, remain inside thedroplets and forms the core of the nanospheres after the solvent isevaporated. PEG chains inside the core should be avoided, because thiscan lead to absorption of water by the core followed by the acceleratedand uncontrolled release of the drugs.

After removing the organic solvent, the injectable particles can beisolated from the aqueous phase by centrifugation. They can later bereadily redispersed in water.

In an alternative embodiment, acetone, methanol, or ethanol and theiraqueous solutions can be used in place of the distilled deionized water.In general, water is preferred because it forces a higher concentrationof poly(alkylene glycol) to the surface of the particle. However,acetone can be used as the precipitating solvent if the hydrophobicpolymer, for example, polyanhydride, is sensitive to water.

In another alternative embodiment, the multiblock copolymer can beblended with a second polymer, for example PLGA-PEG mixed with PLGA orPLA, prior to fabrication into the injectable particles, to providedifferent properties on the injectable particles, for example, alteringtheir half-life in vivo. Adding PLGA-PEG to other polymers can increasethe in vivo half-life of the particles.

In a typical embodiment, the second polymer can be mixed with themultiblock copolymer in a ratio of greater than 0 up to 100 (percent byweight) and optimally, between one and 30 percent by weight.

The substance to be delivered can be mixed with the copolymer orcopolymer blend in a ratio of greater than 0 to 99, and more preferably,in a ratio of 1 to 70.

Characterization studies were carried out at different drug loadings toinvestigate encapsulation properties and morphological characteristicsof PEG-polyanhydride and PEG-polyester nanospheres. Particle size wasmeasured by quasi-elastic light scattering (QELS). The instruments usedwere a Lexel Argon-ion laser (Fremont, Calif., U.S.A.) (model BI-200SM),with a Brookhaven apparatus consisting of a goniometer and a 136 channeldigit correlator and a signal processor. Measurements were made with alaser at a wavelength of 488 nm at a scattering angle of 90°. The imageof the nanospheres was taken by atomic force microscopy (AFM). Theapparatus (Nanoscope III, Digital Instruments, Santa Barbara, Calif.,U.S.A.) consisted of a cantilever oscillating vertically (tapping mode)with a frequency of 350 kHz.

Chemical surface analysis (XPS) was performed to check for the presenceof PEG on the nanospheres surface, and to investigate the presence ofdrug molecules located on the surface. Data were collected by MgKαx-rayswith a power of 300 W on a Perkin-Elmer 5100 apparatus.

To check polymer degradation, lactic acid was detected by colorimetricmethod using Lactate Reagent (Sigma) for a quantitative determination oflactate at 540 nm.

Differential scanning calorimetry (DSC) was performed to detect drugcrystallization inside the nanospheres and to investigate any possibleinteraction between the drug and the polymer.

Morphological analysis of the nanosphere inner core was carried out bytransmission electron microscopy of a cross-section of samples obtainedby freeze fracture.

Drug loading was measured by dissolving lyophilized nanospheres into anappropriate solvent and assaying the amount of drug (lidocaine orprednisolone) spectrophotometrically.

PEG-coated nanospheres are examples of preferred nanospheres, and can beprepared from multiblock copolymers formed by covalently linking amultifunctional compound with at least one poly(ethylene glycol) (PEG)and at least one hydrophobic bioerodible polymer, such as a polyester,for example, (poly(D,L lactic acid), or poly(lactic co-glycolic acid), apolylactone such as ε-polycaprolactone) or a polyanhydride, such as(poly(sebacic acid).

Light scattering studies have indicated that the size of the resultingparticles can be determined by the viscosity of the organic phase, ratioof organic to aqueous phase, and sonication power and time. Increasedviscosity yields bigger particles and a higher ratio of the aqueousphase volume as compared to organic phases yields smaller particles. Anexample of the effect of the sonication power and time is as follows: 25mg polymer/2 ml CH₂ Cl₂ is added to 30 ml of 0.3% polyvinyl alcoholsolution. The mixture is vortexed for 30 seconds at the maximum strengthand then sonicated by probe sonicator for 30 seconds at the output 7.The conditions can reproducibly yield nanoparticles. These parameterscan be optimized to obtain nanospheres having desired size range with anarrow unimodal size distribution.

Using non-linear block copolymers, the density of the hydrophilic blockat the nanosphere surface can be increased and blood circulation ofthese carriers can be prolonged, relative to using a linear copolymer.When multiblock copolymers containing multiple PEG blocks are used,there is typically more PEG on the surface of nanospheres prepared frombrush copolymers than on the surface of nanospheres prepared from linearcopolymers, as shown by ESCA. The amount of PEG (deducted from the ratiobetween PEG and PLA or PLGA comparing C peaks convolution) can beincreased from 35.65% to more than 44% using non-linear multiblockcopolymers as compared with linear copolymers.

Other characterization studies were carried out to investigatemorphological characteristics and encapsulation properties ofPEG-polyanhydride and PEG-polyester nanospheres, at different drugloadings. Cross-section images of freeze-fractured nanospheres wereobtained by TEM, showing the particle dense core. Partial drugrecrystallization was shown by DSC data.

The chemical composition of the nanosphere can be important to thedetermination of the final particle size. Nanospheres prepared frommultiblock brush copolymers that include a significant amount of PEG onthe surface of the particle are typically in the size range of 180 nm orgreater. The diameter can increase up to 240 nm in the case of thehighest PEG m.w. in (PEG 20K)₃ -PLA particles, in contrast to PLAnanoparticles, where the diameter can be less than 120 nm. Surprisingly,this is in contrast to particles prepared from linear copolymers, suchas PEG-PLGA particles, in which the PEG in PEG-PLGA particles was ableto reduce nanosphere size, as compared to not-coated particles. Thecomposition of the hydrophobic block(s) also affects the particle size.For example, using polycaprolactone, which is more soluble in methylenechloride, to form the nanosphere core, particles with a diameter of lessthan 100 nm can be obtained. Drug loading appears to have little effecton particle size. Particles loaded with lidocaine and prednisolone canshow the same size even when the amount of drug loaded is as high as45%.

Preparation of Microparticles

Microparticles can be prepared using the methods as described above forpreparing nanoparticles, without using an ultrasonic bath. Themicroparticles can also be prepared by spraying a solution of themultiblock copolymer in organic solvent into an aqueous solution.

Composition of Particles

As described above, particles are formed from multiblock copolymersprepared by covalently linking a multifunctional compound with at leastone hydrophilic polymer, such as a poly(alkylene glycol) with amolecular weight of between 500 and 20,000 or a polysaccharide moiety,and at least one hydrophobic polymer, such as poly(lactic-co-glycolicacid), poly(lactic acid), poly(glycolic acid), polyanhydride,polyphosphazene, polycaprolactone or other biodegradable, biocompatiblepolymers. The multifunctional compound can be substituted with betweenone and ten hydrophilic polymers and between one and ten hydrophobicpolymers.

As used herein, a hydrophilic polymer refers to a polymer that absorbsor adsorbs water. Preferred molecular weight ranges are between 500 and20,000.

As used herein, a hydrophobic bioerodible polymer refers to a polymerthat does not absorb or adsorb water. Preferred molecular weight rangesare between 500 and 20,000.

As used herein, a polysaccharide refers to a carbohydrate composed ofmany monosaccharides.

As used herein, a multifunctional compound refers to a compound with atleast two functional groups capable of being coupled with functionalgroups on a polymer. The compound can be a linear, branched or cyclicalkyl group, an aromatic group, a heterocyclic group, or a combinationthereof. The types of groups include but are not limited to hydroxyl,thiol, amino, carboxylic acid, aldehyde, sulfonic acid, and phosphoricacid groups. Preferably, the compound is non-toxic and biodegradable.Examples of preferred multifunctional compounds include, but are notlimited to, tartaric acid, mucic acid, citric acid, and tri, tetra-andpolycarboxylic acids, including benzene tetracarboxylic acid, and tri,tetra and polyalcohols, and molecules with combinations of carboxyl andhydroxyl groups.

Size of Particles

As described herein, the typical size of the particles is between 50 nmand 1000 nm, preferably between 90 nm and 200 nm. Although themethodology produces particles between 50 and 1000 nm, or nanoparticles,it is possible to increase the diameter of the resulting particles toform microparticles having a diameter of 1 micron or greater, asdescribed above. For ease of reference herein in the generaldescriptions, both microparticles and nanoparticles will be referred toas particles unless otherwise specified.

As used herein, the term nanoparticle refers to a solid particle of sizeranging from 10 to 1000 nm. The `ideal` nanoparticle is biodegradable,biocompatible, has a size of less than 200 nm and has a rigidbiodegradable core into which a substance to be delivered can beincorporated.

The term "microparticle," as used herein, refers to a particle of sizeranging from one or greater up to 1000 microns.

The nanoparticles specifically described herein can be fabricated asmicroparticles if more appropriate for the desired application.

Structure of Particles.

FIGS. 1a, 1b and 1c are schematic representations of embodiments of ananoparticle prepared as described herein. FIG. 1a, the particle has abiodegradable solid core 12 containing a biologically active material14, and one or more poly(alkylene glycol) moieties 16 on the surface.The surface poly(alkylene glycol) moieties 16 have a high affinity forwater that reduces protein adsorption onto the surface of the particle.The recognition and uptake of the nanoparticle by thereticulo-endothelial system (RES) is therefore reduced. The terminalhydroxyl group of the poly(alkylene glycol) can be used to covalentlyattach biologically active molecules, as shown in FIG. 1b, or moleculesaffecting the charge, lipophilicity or hydrophilicity of the particle,onto the surface of the nanoparticle. In FIG. 1c, the PEG is a branchedshorter chain PEG molecule than in FIG. 1a.

A nanosphere refers to a nanoparticle that is spherical in shape. Theshape of the nanoparticles prepared according to the procedures hereinor otherwise known is easily determined by scanning electron microscopy.Spherically shaped nanoparticles are preferred for circulation throughthe bloodstream. If desired, the particles can be fabricated using knowntechniques into other shapes that are more useful for a specificapplication.

Degradation Properties.

The term biodegradable or bioerodible, as used herein, refers to apolymer that dissolves or degrades within a period that is acceptable inthe desired application (usually in vivo therapy), usually less thanfive years, and preferably less than one year, on exposure to aphysiological solution with a pH between 6 and 8 having a temperature ofbetween 25 and 37° C. In a preferred embodiment, the nanoparticledegrades in a period of between 1 hour and several weeks, depending onthe desired application.

Copolymers for the Construction of Injectable Nanospheres

The period of time of release, and kinetics of release, of the substancefrom the nanoparticle will vary depending on the copolymer or copolymermixture or blend selected to fabricate the nanoparticle. Given thedisclosure herein, those of ordinary skill in this art will be able toselect the appropriate polymer or combination of polymers to achieve adesired effect.

III. Substances to be Incorporated Onto or Into Particles

Materials to be delivered

A wide range of biologically active materials or drugs can beincorporated onto or into the particles. The substances to beincorporated should not chemically interact with the polymer duringfabrication, or during the release process. Additives such as inorganicsalts, BSA (bovine serum albumin), and inert organic compounds can beused to alter the profile of substance release, as known to thoseskilled in the art. Biologically-labile materials, for example,procaryotic or eucaryotic cells, such as bacteria, yeast, or mammaliancells, including human cells, or components thereof, such as cell walls,or conjugates of cellular can also be included in the particle. The termbiologically active material refers to a peptide, protein, carbohydrate,nucleic acid, lipid, polysaccharide or combinations thereof, orsynthetic inorganic or organic molecule, that causes a biological effectwhen administered in vivo to an animal, including but not limited tobirds and mammals, including humans. Nonlimiting examples are antigens,enzymes, hormones, receptors, and peptides. Examples of other moleculesthat can be incorporated include nucleosides, nucleotides, antisense,vitamins, minerals, and steroids.

Injectable particles prepared according to this process can be used todeliver drugs such as nonsteroidal anti-inflammatory compounds,anesthetics, chemotherapeutic agents, immunotoxins, imunosuppressiveagents, steroids, antibiotics, antivirals, antifungals, and steroidalantiinflammatories, anticoagulants. For example, hydrophobic drugs suchas lidocaine or tetracaine can be entrapped into the injectableparticles and are released over several hours. Loadings in thenanoparticles as high as 40% (by weight) have been achieved. Hydrophobicmaterials are more difficult to encapsulate, and in general, the loadingefficiency is decreased over that of a hydrophilic material.

In one embodiment, an antigen is incorporated into the nanoparticle. Theterm antigen includes any chemical structure that stimulates theformation of antibody or elicits a cell-mediated humoral response,including but not limited to protein, polysaccharide, nucleoprotein,lipoprotein, synthetic polypeptide, or a small molecule (hapten) linkedto a protein carrier. The antigen can be administered together with anadjuvant as desired. Examples of suitable adjuvants include syntheticglycopeptide, muramyl dipeptide. Other adjuvants include killedBordetella pertussis, the liposaccharide of Gram-negative bacteria, andlarge polymeric anions such as dextran sulfate. A polymer, such as apolyelectrolyte, can also be selected for fabrication of thenanoparticle that provides adjuvant activity.

Specific antigens that can be loaded into the nanoparticles describedherein include, but are not limited to, attenuated or killed viruses,toxoids, polysaccharides, cell wall and surface or coat proteins ofviruses and bacteria. These can also be used in combination withconjugates, adjuvants, or other antigens. For example, Haemophiliusinfluenzae in the form of purified capsular polysaccharide (Hib) can beused alone or as a conjugate with diptheria toxoid. Examples oforganisms from which these antigens are derived include poliovirus,rotavirus, hepatitis A, B, and C, influenza, rabies, HIV, measles,mumps, rubella, Bordetella pertussus, Streptococcus pneumoniae, C.diptheria, C. tetani, Cholera, Salmonella, Neisseria, and Shigella.

Non-pharmaceutical uses for the injectable particles include delivery offood additives, including stabilizers and dispersants or other viscositymodifying agents, controlled and selective delivery of pesticides,herbicides, insecticides, fertilizer, and pheromones, and in color andink formulations in the printing and ink industry.

Incorporation of Substances for Diagnostic Purposes.

In another embodiment, a gamma-labelled injectable nanoparticle isprovided that can be used to monitor the biodistribution of theinjectable particle in vivo. Any pharmaceutically acceptablegamma-emitting moiety can be used, including but not limited to indiumand technetium. The magnetic particles can be prepared as describedherein, or alternatively, magnetic nanoparticles, includingsurface-modified magnetic nanoparticles can be purchased commercially,the surface further modified by attaching the hydrophilic polymericcoating.

For example, the magnetic nanoparticle can be mixed with a solution ofthe hydrophilic polymer in a manner that allows the covalent binding ofthe hydrophilic polymer to the nanoparticle. Alternatively, agamma-emitting magnetic moiety is covalently attached to the hydrophilicor hydrophobic bioerodible polymeric material of the particle. Thelarger the size of the magnetic moiety, the larger the size of theresulting particles obtained.

Other materials can also be incorporated into the injectable particlesfor diagnostic purposes, including radiopaque materials such as air orbarium and fluorescent compounds. Hydrophobic fluorescent compounds suchas rhodamine can be incorporated into the core of the injectableparticles. Hydrophilic fluorescent compounds can also be incorporated,however, the efficiency of encapsulation is smaller, because of thedecreased compatibility of the hydrophobic biodegradable core with thehydrophilic material. The hydrophilic material must be dissolvedseparately in water and a multiple emulsion technique used forfabrication of the particle.

In one embodiment, the particles include a substance to be delivered anda multiblock copolymer that is covalently bound to a biologically activemolecule, for example, an antibody or antibody fragment, such as the Fabor Fab₂ antibody fragments, wherein the particle is prepared in such amanner that the biologically active molecule is on the outside surfaceof the particle.

Modification of Surface Properties of Particles.

The charge, lipophilicity or hydrophilicity of the particle can bemodified by attaching an appropriate compound to the hydrophilic polymeron the surface of the particle. The particle can also be coated with adextran, which are in general more hydrophilic than poly(alkyleneglycol) but less flexible. Dextran coated nanoparticles are useful formagnetic resonance imaging (MRI).

Attachment of specific ligands to particle surfaces.

The injectable particles prepared as described herein can be used forcell separation, or can be targeted to specific tissues, by attaching tothe surface of the particle specific ligands for given cells in amixture of cells. When magnetic particles are also incorporated, theparticles can be targeted using the ligands, such as tissue specificreceptors or antibodies to tissue specific surface proteins, thenmaintained at the targeted cells using a magnetic field while theparticles are imaged or a compound to be delivered is released.

For example, in one embodiment, carmustine (BCNU) or other anti-canceragent such as cis-platin is incorporated in the core of the injectableparticles and antibodies to the target cancerous cells are covalentlybound to the surface of the injectable particle.

Pharmaceutical Administration of Nanospheres

The injectable particles described herein can be administered to apatient in a variety of routes, for example, orally, parenterally,intravenously, intradermally, subcutaneously, or topically, in liquid,cream, gel or solid form.

The particles can be lyophilized and then formulated into an aqueoussuspension in a range of microgram/ml to 100 mg/ml prior to use.Alternatively, the particles can be formulated into a paste, ointment,cream, or gel, or transdermal patch.

The nanoparticle should contain the substance to be delivered in anamount sufficient to deliver to a patient a therapeutically effectiveamount of compound, without causing serious toxic effects in the patienttreated. The desired concentration of active compound in thenanoparticle will depend on absorption, inactivation, and excretionrates of the drug as well as the delivery rate of the compound from thenanoparticle. It is to be noted that dosage values will also vary withthe severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions.

The particles can be administered once, or may be divided into a numberof smaller doses to be administered at varying intervals of time,depending on the release rate of the particle, and the desired dosage.

IV. Coatings of Implantable Devices

Polymers loaded as described herein can also be used to coat implantabledevices, such as stents, catheters, artificial vascular grafts, andpacemakers. The device can be coated with the lyophilized powder of theinjectable particles, or otherwise as known to those skilled in the art.The coating can release antibiotics, anti-inflammatories, oranti-clotting agents at a predetermined rate, to prevent complicationsrelated to the implanted devices. Controlled delivery devices preparedas described herein can also be used as ocular inserts for extendedrelease of drugs to the eye.

EXAMPLES

The preparation of specific multiblock copolymers of hydrophobicbioerodible polymers such as PLA and PLGA, and hydrophilic polyalkyleneglycols such as PEG, with multifunctional compounds such as tartaricacid, mucic acid, citric acid, benzene tetracarboxylic acid, gluconicacid, and butane diglycidyl ether are described in detail below. Thesepolymers were prepared with PEG of various chain lengths, and withvarious hydrophobic polymers. Given this detailed description, one ofskill in the art will know how to produce a wide variety of multiblockcopolymers suitable for fabrication into injectable nanospheres.

Materials and Methods.

Low toxicity stannous octoate was purchased from ICN. D,L-lactide waspurchased from Aldrich Chemical Company, and glycolide fromPolysciences, Inc. These compounds were recrystallized before use fromethyl acetate. High purity monomethoxy PEG (M-PEG) with molecular weight5,000, 12,000 and 20,000 was purchased from Shearwater Polymers, Inc.The number average molecular weight of the polymer was determined withon a Perkin-Elmer GPC system with an LC-25 refractive index detectorequipped with a mixed bed Phenogel column filled with 5 μm particlesfrom Phenomenex. Chloroform was used as the eluent, with a flow rate of0.9 ml/min. The molecular weights were determined relative to narrowmolecular weight polystyrene and poly(ethylene glycol) standards fromPolysciences.

Thermal transition data was collected with a Perkin-Elmer DSC-7 (NewtonCenter, Mass.). The sample weight ranged from 20 to 25 mg. Indium wasused for temperature and enthalpy calibrations. Each sample wassubjected to a heat-cool-heat cycle from -60 to 150° C. with a rate of10° C./min. Wide angle x-ray diffraction spectra were obtained with aRigaku Rotaflex Diffractometer from Rigaku Corporation (Danvers, Mass.)with S=0.05 using a Nickel filtered Cu Kαsource. The data was analyzedon a Micro Vax II computer. The IR spectra were recorded on a Nicolet500 spectrometer using a polymer powder melted on sodium chloridecrystals to obtain thin films. ¹³ C NMR studies were conducted onsamples dissolved in deuterated chloroform with a Nicolet NT-360spectrometer. Peak fitting was carried out with a VG data system.

Example 1 Synthesis of (methoxy-PEG-NH₂)₃, citrate (compound A)

Three PEG citrates were prepared as follows: 1. PEG-NH₂ (1 gram,MW=5,000, Sherewater) was reacted with citric acid (14 mg, 0.33equivalents) using dicyclohexylcarbodiimide (DCC) (54 mg, 1 equivalent)and DMAP (4 mg, catalyst) in 10 ml of dry dichloromethane. The reactionwas continued for 2 days at room temperature with magnetic stirring. TheDCU by-product was isolated by filtration and the filtrate was pouredinto 100 ml of ether:petroleum ether 1:1 mixture. The precipitatedpolymer was washed with ether and dried to yield 0.8 grams of a whitepowder. The product did not contain acid groups (Bromophenol test) andshowed a single peak at the GPC chromatogram in the area of 15,000. IRshowed typical ester peak (1720 cm⁻¹). Methoxy-PEG citrate trimers withPEG of the following molecular weights, 1,900; 12,000; and 20,000 wereprepared using this procedure.

The PEG derivatives of tartaric acid [(methoxy-PEG) 2-tartrate], mucicacid [(methoxy-PEG)-2-mucoate], and gluconic acid (methoxy-PEG-mucoate)with various PEG chain length were prepared similarly. All derivativespossessed the appropriate molecular weight (determined by GPC using PEGstandards), showed a negative result in the bromophenol test forcarboxylic acids, and had an absorption peak at 1720 typical for amidebonds.

Example 2 Esterification reaction between methoxy PEG-OH and citric acidusing DCC

The reaction conditions were the same as above, and an 80% conversionwas obtained, as determined by GPC (compound A-1).

Example 3 Direct esterification reaction between methoxy PEG-OH andcitric acid.

In a 100 ml round bottom flask equipped with a Dean-Stark azeotropeapparatus, methoxy PEG-OH (MW 1900, Polysciences) was reacted withcitric acid (0.33 equivalents) in toluene and sulfuric acid as catalyst(1%). The reaction was conducted under reflux using azeotrope for H₂ Oremoval. About 75% yield was obtained as determined by GPC.

Example 4 Trans esterification reaction between methoxy PEG-OH andmethyl citrate ester.

Citrate methyl ester was obtained from the reaction between citric acidand access methanol at reflux.

The resulting trimethyl citrate (1 equivalent) was reacted with methoxyPEG Mw-1900 (3 equivalents) in refluxing toluene for three hours. Theproduct was isolated in about 70% yield, as determined by GPC, afterevaporation of the toluene and extraction with diethyl ether.

Example 5 Synthesis of (PEG)₃ -citrate-polylactide [PEG₃ -PLA] or PEG₃-caprolactone [PEG₃ -PCL] diblock copolymers (compound A1, FIG. 2a )

PEG₃ -citrate (1 gram) (Sherewater, MW-5,000, 12,000, and 20,000) wasdissolved in 20 ml benzene. Lactide (5 grams) (Aldrich, 99%+) was addedand the solution was allowed to reflux and azeotrope for 60 min.Stannous octoate (0.2% by weight (per lactide)) was added as a 1%solution in benzene. The reaction was refluxed for 5 hours, the solventwas removed azeotropically and a viscous material was obtained. Thepolymerization was continued for 2 hours at 130° C. The resultingpolymer was a clear, slightly yellow mass, and showed a high molecularweight (Table 1). The multiblock copolymers of PEG-polycaprolactone weresimilarly synthesized. The polymers were soluble in common organicsolvents.

                  TABLE 1                                                         ______________________________________                                        Molecular weights of PEG block copolymers                                     Polymer             Mn      Mw       MP                                       ______________________________________                                        PEG-PCL block copolymers                                                      PCL-PEG 5k (1:5 w/w)                                                                              29,500   70,100  55-58                                    PCL-PEG 12k (1:5 w/w)                                                                             25,500   88,100  55-58                                    PCL-PEG 20k (1:5 w/w)                                                                             34,500  105,900  50-56                                    PEG-citrate-PLA multiblock                                                    copolymers                                                                    (PEG-NH 5k).sub.3 citrate                                                                         15,600  --                                                (PEG-NH 5k).sub.3 citrate-PLA                                                                     71,900  228,100  65-75                                    (PEG-NH 5k).sub.3 citrate-PCL                                                                     42,000  170,000  52-58                                    PSA-PEG block copolymers                                                      PSA-COO-PEG 5k (3:1 w/w)                                                                          19,500  119,100  65-78                                    PSA-COO-PEG 12k (3:1 w/w)                                                                         21,000  144,200  65-78                                    PSA-COO-PEG 20k (3:1 w/w)                                                                         18,500  105,000  65-78                                    P(SA-PEG 5k) random block (1:3)                                                                   21,000  105,400  64-73                                    PSA                 17,200   81,300  80-82                                    P(FAD)-COO-PEG 5k (3:1)                                                                           12,000   34,000  42-48                                    P(CPP-SA)1:1-COO-PEG 5k (3:1)                                                                     12,000   34,000  42-48                                    ______________________________________                                    

Example 6 Synthesis of multiblock (brush) PEG-ligand-PLA (compound B,FIG. 2a

Citric acid (0.1 mole) was reacted with a mixture of methoxy-PEG amine(MW 1900) (0.2mole) and benzyl ester carboxy-PEG-amine (MW 5,000) (0.1mole) using DCC (0.33 equivalents) and DMAP (0.01 mole, catalyst) in 100ml of dry dichloromethane. The reaction was continued for 2 days at roomtemperature with magnetic stirring. The DCU by-product was isolated byfiltration and the filtrate was poured into 500 ml of ether:petroleumether 1:1 mixture. The precipitated polymer was washed with ether anddried to yield a white powder in 90% yield. The product did not containacid groups (Bromophenol test) and showed a single peak at the GPCchromatogram with a molecular weight of 9,000. IR showed typical esterpeak (1720 cm⁻¹). Block copolymers with lactide and caprolactone weresynthesized using the same method described for PLA-PEG brush blockcopolymers.

The PLA-PEG citrate trimer was dissolved in tetrahydrofuran andhydrogenated with Hydrogen-Palladium catalysis to remove the benzylicprotecting group at the PEG 5000 chain. The end chain carboxylic acidPEG was then reacted with bovine serum albumin (representing a ligand)using DCC as an activating agent for amide coupling.

Similarly, two or three ligands can be attached to (PEG) 3-citrate byusing two or three equivalents of benzyl carboxylate-terminatedPEG-amines, using the above method.

Example 7 Preparation of PEG₂ -tartrate-PLA₂ (Compound C, FIG. 2b)

Di-PEG tartrate was prepared from the reaction between amino terminatedmethoxy PEG and tartaric acid with DCC as the activating agent, usingthe procedure described for the synthesis of (PEG)₃ -citrate. The di-PEGtartrate derivative was reacted with lactide or glycolide mixtures toform clear polymers (Table 1).

Example 8 Preparation of di-methoxy PEG-mucoate-tetra PLA (Compound D,FIG. 2b)

Mucic acid (Aldrich) was reacted with two equivalents of methoxy PEG inthe presence of DCC in DMF to form di-PEG-mucoate which wascopolymerized with lactide, glycolide or caprolactone to form highmolecular weight (Mw=65,000-95,000) hexa-armed block copolymers.

Example 9 Preparation of penta-methoxy PEG-glucoronate-anhydride(Compound E, FIG. 2b)

Gluconic acid was reacted with carboxylic acid terminated methoxy PEG(MW=5,000, Sherewater) in the presence of DCC to form (PEG)₅ -gluconate.The penta-PEG compound was polymerized with sebacic acid (1:5 weightratio) using acetic anhydride as a dehydrating agent. Polymers with amolecular weight of approximately 75,000 were obtained.

Example 10 Preparation of mono-PEG-penta PLA glucoronate (Compound F,FIG. 2b)

Gluconic acid was reacted with amino terminated methoxy PEG (MW=5,000,Sherewater) in the presence of DCC in dichloromethane of DMF to formPEG-gluconate amide. The gluconate PEG derivative was polymerized withlactide, glycolide or caprolactone (1:5 weight ratio).

Example 11 Preparation of PEG-di-PLA (compound G, FIG. 2c)

Methoxy-PEG-epoxide terminated (Sherewater) was hydrolyzed in a sodiumcarbonate solution overnight at room temperature. The resulted PEG withtwo hydroxyl groups was isolated by precipitation in ether:methanol 1:1mixture and dried. The dihydroxy-terminated PEG was block copolymerizedwith lactide, glycolide and caprolactone to form high molecular weightpolymers (The molecular weight was in the range of 70,000 to 115,000).

Example 12 Preparation of trimethoxy PEG-citrate-poly(sebacic anhydride)diblock copolymer (Compound H, FIG. 2a)

Trimethoxy-PEG-citrate (0.01 mole, prepared as above) reacted withaccess adipoyl chloride (0.012 mole) in dichloromethane withtriethylamine as a proton acceptor. After 24 hours at room temperature,water was added, the reaction mixture was stirred at room temperaturefor one hour, and the polymer was isolated by the adding a mixture ofmethanol-diethyl ether 1:1. The resulting trimethoxy-PEG-citrate-adipatewas reacted with acetic anhydride to form the acetate anhydridederivative, which was polymerized with a sebacic anhydride prepolymer toform a multiblock copolymer with a molecular weight of Mw=58,000;Mn=31,000. MP=65°-74° C.

Example 13 Benzene tetracarboxylic anhydride (BTCA) derivatives(Compound I, FIG. 2d)

BTCA was reacted with two equivalents of methoxy PEG amine in refluxingTHF for 5 hours to yield dimethoxy-PEG tetracarboxybenzoate, with tworemaining carboxylic groups. The PEG-dimer was reacted with aceticanhydride and then with sebacic anhydride to form the tetra-armeddiblock PEG₂ -benzene-PSA₂.

Alternatively, polycaproiactone diol (Mw=3,000, Polysciences) wasreacted with dimethoxy-PEG tetracarboxybenzoate containing 2 carboxylicacids to form the tetra-armed PEG-PCL diblock copolymer. The PLA or PCLblock copolymers were prepared, and then the carboxylic acid groups ofthe PEG-benzene tetracarboxylate were reacted With propylene oxide toform the hydroxyl derivative available for the block copolymerizationwith lactide, glycolide and caprolactone.

Example 14 Butane diglycidyl ether based tetra-arm diblock copolymers(Compound J, FIG. 2e)

Butane diglycidyl ether was reacted with two equivalents ofmethoxy-PEG-OH in refluxing THF for 10 hours. The PEG dimer was blockcopolymerized with lactide, glycolide or caprolactone in toluene withstannous octoate as catalyst. High molecular weight polymers wereobtained (Please define high molecular weight).

Example 15 Multiblock copolymers based on the 1,4;3,6-dilactone ofglucaric acid (Compound K, FIG. 2f)

PLA was polymerized in the presence of the dilactone (5:1 weight ratio)using stannous octoate as catalyst in benzene. The two carboxylic acidgroups were used to attach methoxy-PEG-amine via an amide bond.

Example 16 Synthesis of PLA-citrate-dextran (Compound N, FIG. 2h)

Dextran, a clinically used biodegradable material, was used asalternative hydrophilic polymer to PEG. The benzyl ester of citric acidwas polymerized with lactide to form a PLA-terminated citrate esterwhich was hydrogenated to remove the benzyl groups. The citric acidterminated-PLA was esterified with dextran to form PLA-citrate-Dextran₃

Example 17 Derivatives of PEG 2-hydroxyadipaldehyde (Compound M, FIG.2i)

2-Hydroxyadipaldehyde (Aldrich) was reacted with amino terminated PEG toform the Schiff base which was hydrogenated with NaBH₄ to form thecorresponding amine. The di-PEG derivative was reacted with lactide orcaprolactone in the presence of stannous octoate to form the PLA orPCL-PEG₂ diblock copolymer.

Example 18 Derivatives of Dextran or Ligand 2-hydroxyadipaldehyde(Compound M-1, FIG. 2h)

2-Hydroxyadipaldehyde is reacted with lactide in the presence ofstannous octoate to form adipaldehyde-terminated PLA. The aldehydegroups are reacted with amino side groups of a ligand (peptide orprotein) to form a di-ligand-PLA diblock. Alternatively, the aldehydicterminals are reacted with ethylene diamine to form PLA-terminated withdiamino groups. This polymer is reacted with an oxidized polysaccharide,such as dextran or amylose, to form a PLA-di-(polysaccharide)derivative.

Example 19 Polyanhydride-PEG

Polyanhydride-terminated PEG was prepared by melt condensing a sebacicacid prepolymer (synthesized by refluxing sebacic acid in aceticanhydride and precipitating the resulting polymer in ether/petroleumether solution) and methoxy PEG-OH or methoxy PEG-carboxylate acetateanhydride. In a typical experiment, methoxy-PEG-carboxylate (1 gram) wasmixed with sebacic acid prepolymer (3 grams). The mixture waspolymerized at 180° C. under vacuum (0.1 mm Hg) for 90 minutes to yieldthe polymer. The polymer showed IR absorption at 1805 and 1740 cm-1(typical for aliphatic anhydride bonds), and the ¹ H-NMR spectrum fitthe polymer structure.

Example 20 Drug Release Characteristics

Lidocaine and prednisolone (Sigma), were selected for encapsulationbecause of their low water solubility, high solubility in organicsolvents and ease of detection by UV spectrophotometry. Release testswere carried out with nanospheres loaded with lidocaine in differentamounts (20% wt, 33% wt), in phosphate buffer solution (PBS, pH 7.4) at37° C. A dialysis membrane (50,000 cut-off) was filled with a suspensionof lyophilized nanospheres (10 mg/5 ml PBS) and then placed into 25 mlof PBS. Samples were taken from the outer solution, then replaced everytime with fresh ones. Drug released was detected spectrophotometricallyat 240 nm.

While high encapsulation efficiency can be achieved with particles madefrom multiblock brush copolymers, it can be difficult to obtain 100%encapsulation efficienty due to the hydrophilicity of the multiblockcopolymers. It was observed that the encapsulation efficiency can beless than 70% for PEG (m.w. of 5, 12, 20 kDa).

In vitro studies were performed to investigate the releasecharacteristics of PEG-coated nanospheres, in particular to study theeffect of the presence of PEG on the nanosphere surface and the effectof the nanosphere core composition (polymer and drug nature, drugloading) on the drug release kinetics. Suspensions of nanospheres wereeasily obtained by redispersing freeze-dried particles in aqueoussolutions by vortexing, without any further additives. Lidocaine wasused as a model drug. The release of lidocaine was studied in particlesmade from linear PEG-PGLA copolymers as well as non-linear brushcopolymers.

Both types of particles show a continuous release in vitro over severalhours, but have different release kinetics. The molecular weight doesnot effect the release pattern of PEG-PLGA nanospheres, since the drugis completely released in about ten hours using copolymers with a PEGm.w. of 5, 12, 20 KDa. The presence of PEG on the surface of thenanospheres is not expected to modify the drug release. However, withmultiblock copolymers, factors such as higher PEG density and PEG chainlength can slow down drug release. In ten hours, more than 90% oflidocaine was released from PLA nanospheres, but only 60% from (PEG20K)₃ -PLA particles.

Drug release from nanospheres made from PEG-ε-polycaprolactone isbiphasic.

Because of polymer erosion, it would ordinarily be expected that a coremade of polyanhydride should lead to a faster drug release. However,after an initial fast release in the first two hours, drug releasereached a plateau, although drug was released at a constant rate for anadditional eight hours.

Polymer degradation kinetics were also investigated in vitro. WithPEG-PLGA, PEG-PCL and (PEG)₃ -PLA particles, the polymers start todegrade after weeks. Nanosphere cores made of polyanhydrides start todegrade immediately. In the first case, drug release is governed by adiffusion mechanism, since the drug can be completely released beforepolymer degradation occurs. With polyanhydrides, polymer erosion affectsdrug release, and drug characteristics have a more important role inrelease kinetics. The particle's small size and large surface areaincreases the rate of polymer erosion relative to other drug deliverysystems, such as slabs, and afterwards drug solubility governs thedissolution kinetics.

The amount of drug loading can have a strong effect on the releasekinetics. PEG-PLGA nanospheres containing 33% wt of lidocaine canrelease the drug for over 12 hours. Surprisingly, particles loaded with10% of the drug can show complete drug release in 6 hours. Increaseddrug loading can cause part of the drug loaded in the core torecrystallize, as shown by DSC. The presence of crystals of ahydrophobic drug, such as lidocaine, can slow down the release kinetics.ESCA studies performed on drug loaded nanospheres confirmed that drugcrystals were not located on the nanosphere surface. The polymercomposition was also modified and the drug loading was increased up to45% wt.

Example 21 Evaluation of Biodistribution of ¹¹¹ In-labeled Nanoparticlesin vivo

Indium 111 ("In") can be directly attached to the multiblock copolymerchains by complex formation. In and diethyltriamiopentaacetic acid(DTPA) are reacted with stearylamine. The resulting compound,In-DTPA-stearylamide, is hydrophobic enough to interact to beencapsulated within the hydrophobic core. In this case, the molecularweights of the hydrophilic and hydrophobic polymers have little effecton the interaction. After incubation at 37° C. in PBS or horse serum formore than 24 hours, label loss can be assessed by measuring theradioactivity of the supernatant solutions after centrifugation. Thislabelling method can therefore be useful for in vivo studies, bygamma-scintography or by direct measurement of the radioactivity in theblood and/or different organs.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of theappended claims.

We claim:
 1. Multiblock copolymers consisting essentially of amultifunctional compound selected from the group consisting of gluconicacid, tartaric acid, mucic acid, citric acid, benzene dicarboxylic acid,benzene tricarboxylic acid, benzene dicarcoxylic acid and butanediglycidyl ether covalently linked with one or more hydrophillicpolymers and one or more hydrophic bioerodible polymers and including atleast three polymer blocks.
 2. The multiblock copolymer of claim 1wherein the hydrophilic polymer is selected from the group consisting ofpolyalkylene glycol, polypyrrolidone, polyvinyl alcohol and dextran. 3.The multiblock copolymer of claim 1 wherein the hydrophobic polymer isselected from the group consisting of polyanhydride, polyphosophaze,polyhydroxybutyric acid, polyorthoesters, polysiloxanes,polycaprolactone, poly(α-hydroxy acids) and copolymers prepared from themonomers of these polymers.
 4. A particle having a diameter of between50 nm and 1000 μm formed of or coated with a multiblock copolymer,consisting essentially of a multifunctional compound covalently linkedwith one or more hydrophilic polymers and one or more hydrophobicbioerodible polymers to form a coblock polymer including at least threepolymer blocks, wherein the hydrophilic polymers are on the surface ofthe particle in an amount effective to decrease uptake of the particlesby the reticuloendothelial system, comprising detectable moleculesselected from the group consisting of substances detectable by x-ray,fluorescence, ultrasound, magnetic resonance imaging and radioactivity.5. The particle of claim 4 further comprising a substance to bedelivered selected from the group consisting of peptides, proteins,carbohydrates, nucleic acids, lipids, polysaccharides, combinationsthereof, and synthetic inorganic or organic molecules that cause abiological effect when administered to an animal.
 6. The particle ofclaim 4 wherein the hydrophilic polymer is selected from the groupconsisting of polyalkylene glycols, polyvinyl alcohols, polypyrrolidonesand dextrans.
 7. The particle of claim 6 wherein the polyalkylene glycolis selected from the group consisting of polyethylene glycol andcopolymers of polyoxyethylene and polyoxypropylene.
 8. The particle ofclaim 4 wherein the hydrophobic polymer is selected from the groupconsisting of polyanhydrides, poly(α-hydroxy acids), polyorthoesters,polyphosphazenes, polysiloxanes, polycaprolactone and copolymersprepared from the monomers of these polymers.
 9. The particle of claim 4wherein the multifunctional compound is selected from the groupconsisting of gluconic acid, tartaric acid, mucic acid, citric acid,benzene dicarboxylic acid, benzene tricarboxylic acid, benzenetetracarboxylic acid and butane diglycidyl ether.
 10. The particle ofclaim 4 comprising molecules covalently bound to the surface of theparticle via reactive groups on the hydrophilic polymer, wherein themolecules are selected from the group consisting of biologically activemolecules, non-biologically active molecules which can be detected,targeting molecules, and molecules affecting the charge, lipophilicityor hydrophilicity of the particle.
 11. The particle of claim 10, whereinthe targeting molecule is selected from the group consisting ofcompounds specifically reactive with a cell surface component,antibodies and antibody fragments.
 12. The particle of claim 4 whereinthe diameter is less than one micron.
 13. The particle of claim 4wherein the diameter is between one and 1000 microns.
 14. The particleof claim 4 wherein the detectable molecule is detectable by ultrasound.15. The particle of claim 6, wherein the poly(alkylene glycol) ispoly(ethylene glycol).
 16. The particle of claim 4 formed of a core of adifferent material than the coblock polymer coating.
 17. A method formaking a multiblock copolymer by covalently linking a multifunctionalcompound selected from the group consisting of gluconic acid, tartaricacid, mucic acid, citric acid, benzene dicarboxylic acid, benzenetricarboxylic acid, benzene tetracarboxylic acid and butane diglycidylether to one or more hydrophilic polymers and one or more hydrophobicbioerodible polymers, wherein the number of polymer blocks is at leastthree.
 18. The method of claim 17 further comprising forming a particlewith a diameter between 50 nm and 1000 μm of the coblock polymer orcoating a particle with a diameter between 50 nm and 1000 μm with thecoblock polymer.
 19. The method of claim 17 wherein the substance to bedelivered is a biologically active substance selected from the groupconsisting of peptides, proteins, carbohydrates, nucleic acids, lipids,polysacccarides, combinations thereof, and synthetic inorganic ororganic molecules that cause a biological effect when administered invivo to an animal.
 20. The method of claim 17 wherein the hydrophilicpolymer is selected from the group consisting of polyalkylene glycols,polyvinyl alcohols, polypyrrolidones and dextrans.
 21. The method ofclaim 17 wherein the hydrophobic polymer is selected from the groupconsisting of polyanhydrides, poly(α-hydroxy acids), polyorthoesters,polyphosphazenes, polysiloxanes, polycaprolactone, and copolymersprepared from the monomers of these polymers.
 22. The method of claim 18further comprising covalently binding to the surface of the particle viathe terminal hydroxyl group of the poly(alkylene glycol) moleculesselected from the group consisting of biologically active molecules,non-biologically active molecules which can be detected, targetingmolecules, and molecules affecting the charge, lipophilicity orhydrophilicity of the particle.
 23. The method of claim 22 furthercomprising targeting the particle for delivery to a specific cell typeby attaching to the surface of the particle a targeting moleculeselected from the group consisting of compounds specifically reactivewith a cell surface component, antibodies and antibody fragments. 24.The method of claim 22 wherein the molecule is a substance detectable byx-ray, fluorescence, magnetic resonance imaging, ultrasound orradioactivity.
 25. The method of claim 17 wherein the hydrophilicpolymer is selected from the group consisting of polyalkylene glycol,polypyrrolidone, polyvinyl alcohol and dextran.
 26. The method of claim17 wherein the hydrophobic polymer is selected from the group consistingof polyanhydride, polyphosophaze, polyhydroxybutyric acid,polyorthoesters, polysiloxanes, polycaprolactone, poly(α-hydroxy acids)and copolymers prepared from the monomers of these polymers.
 27. Themethod of claim 17 wherein the multifunctional compound is selected fromthe group consisting of gluconic acid, tartaric acid, mucic acid, citricacid, benzene dicarboxylic acid, benzene tricarboxylic acid, benzenetetracarboxylic acid and butane diglycidyl ether.
 28. A method forimaging a patient comprising administering to the patient a particlehaving a diameter of between 50 nm and 100 μm formed of or coated with amultiblock copolymer consisting essentially of a multifunctionalcompound with one or more hydrophilic polymers and one or morehydrophobic bioerodible polymers to form a block copolymer including atleast three polymer block, wherein the hydrophilic polymers are on thesurface of the particle in an amount effective to decrease uptake of theparticles by the reticuloendothelial system, and the particles comprisedetectable molecules selected from the group consisting of substancesdetectable by x-ray, fluorescence, ultrasound, magnetic resonanceimaging and radioactivity.
 29. The method of claim 28 wherein thehydrophilic polymer is selected from the group consisting ofpolyalkylene glycol, polypyrrolidone, polyvinyl alcohol and dextrans.30. The method of claim 29 wherein the polyalkylene glycol is selectedfrom the group consisting of polyethylene glycol and copolymers ofpolyoxyethylene and polyoxypropylene.
 31. The method of claim 28 whereinthe hydrophobic polymer is selected from the group consisting ofpolyanhydrides, poly(α-hydroxy acids), polyorthoesters,polyphosphazenes, polysiloxanes, polycaprolactone, and copolymersprepared from the monomers of these polymers.
 32. The method of claim 28wherein the multifunctional compound is selected from the group groupconsisting of gluconic acid, tartaric acid, mucic acid, citric acid,benzene dicarboxylic acid, benzene tricarboxylic acid, benzenetetracarboxylic acid and butane diglycidyl ether.